The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.     

The Nonphosphorylative Entner-Doudoroff Pathway in the Thermoacidophilic Euryarchaeon Picrophilus torridus Involves a Novel 2-Keto-3-Deoxygluconate- Specific Aldolase

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-¹³C]- and [3-¹³C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.Comparative analyses of sugar-degrading pathways in members of the domain Archaea revealed that all species analyzed so far degrade glucose and glucose polymers to pyruvate via modification of the classical Embden-Meyerhof (EM) and Entner-Doudoroff (ED) pathways found in bacteria and eukarya. Modified EM pathways were reported for hyperthermophilic archaea, including, e.g., the strictly fermentative Thermococcales and Desulfurococcales, the sulfur-reducing Thermoproteus tenax, and the microaerophilic Pyrobaculum aerophilum. These pathways differ from the classical EM pathway by the presence of several novel enzymes and enzyme families, catalyzing, e.g., the phosphorylation of glucose and fructose-6-phosphate, isomerization of glucose-6-phosphate, and oxidation of glyceraldehyde-3-phosphate (18, 22, 25).Modified ED pathways have been proposed for aerobic archaea, including halophiles, and thermoacidophilic crenarchaea, such as Sulfolobus species, and the euryarchaea Thermoplasma acidophilum and Picrophilus torridus. The anaerobic Thermoproteus tenax, which degrades glucose predominantly via a modified EM pathway, also utilizes—to a minor extent (<20%)—a modified ED pathway for glucose degradation. The following ED pathway modifications have been reported in archaea (25). A semiphosphorylative ED pathway was reported in halophilic archaea. Accordingly, glucose is converted to 2-keto-3-deoxy-6-gluconate (KDG) via glucose dehydrogenase and gluconate dehydratase. KDG is then phosphorylated by KDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is split by KDPG aldolase to pyruvate and glyceraldehyde-3-phosphate (GAP). GAP is further converted to form another pyruvate via common reactions of the EM pathway, i.e., phosphorylative GAP dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. The net ATP yield of this pathway is 1 ATP/mol glucose.From initial enzyme studies of the thermoacidophilic archaea Sulfolobus solfataricus, Thermoplasma acidophilum, and Thermoproteus tenax, a nonphosphorylative ED pathway was proposed (25). In this modification of the ED pathway, glucose is converted to KDG via glucose dehydrogenase and gluconate dehydratase, as in the semiphosphorylative pathway, but then the steps differ as follows: KDG is cleaved into pyruvate and glyceraldehyde via 2-keto-3-deoxygluconate-specific aldolase (KDGA). The subsequent oxidation of glyceraldehyde to glycerate involves either NAD(P)⁺-dependent dehydrogenases or oxidoreductases. Glycerate is then phosphorylated by a specific kinase to 2-phosphoglycerate, which is finally converted to pyruvate via enolase and pyruvate kinase. This modification of the ED pathway was called “nonphosphorylative” since it is not coupled with net ATP synthesis.However, recent comparative genomic studies and refined enzyme analyses suggest that the crenarchaea Sulfolobus and Thermoproteus utilize a so-called branched ED pathway, in which a semiphosphorylated route is simultaneously operative in addition to the nonphosphorylative route (25, 32). Accordingly, the semiphosphorylated route involves—via KDG kinase—the phosphorylation of KDG to KDPG, which is then cleaved to pyruvate and GAP by means of a bifunctional KDG/KDPG aldolase, KD(P)GA. GAP is then converted to another pyruvate via nonphosphorylative GAP dehydrogenase (GAPN), phosphoglycerate mutase, enolase, and pyruvate kinase. The net ATP yield of the branched ED pathway is zero. In support of this pathway, the genes encoding gluconate dehydratase, bifunctional KD(P)GA, KDG kinase, and GAPN were found to be clustered in Sulfolobus solfataricus (see Discussion) and Thermoproteus tenax. The key enzyme of the proposed branched ED pathway is the bifunctional KD(P)GA, which catalyzes the cleavage of KDG to pyruvate and glyceraldehyde and cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. This bifunctional aldolase, which has been characterized from S. solfataricus, was found to be identical to a previously described KDG aldolase of the same organism; however, its catalytic property to also utilize KDPG as a substrate has been recognized only recently. In fact, the bifunctional KD(P)GA showed a higher catalytic efficiency for KDPG than for KDG (1, 14). Crystal structures of bifunctional KD(P)GAs of S. solfataricus and T. tenax have been reported (16, 27, 30; G. Taylor [United Kingdom], unpublished data).The branched ED pathway in S. solfataricus has been reported to be promiscuous and therefore represents an equivalent degradation route for both glucose and its C-4 epimer, galactose. Accordingly, glucose dehydrogenase, gluconate dehydratase, KDG kinase, and bifunctional KD(P)GA were found to catalyze the conversion of both glucose and galactose and the corresponding subsequent intermediates, i.e., gluconate/galactonate, KDG/KDGal (KDGal stands for 2-keto-3-deoxygalactonate), and KDPG/KDPGal (KDPGal stands for 2-keto-3-deoxy-6-phosphogalactonate) (4, 12-14).In contrast to crenarchaea, the modified ED pathway in the thermoacidophilic euryarchaea Thermoplasma acidophilum and Picrophilus torridus has not been studied in detail. Enzyme measurements in cell extracts and the characterization of few enzymes suggest the operation of a nonphosphorylative ED pathway in these organisms (2, 3, 17, 19, 25). However, in vivo evidence for the operation of an ED-type pathway, e.g., by ¹³C-labeling experiments with growing cultures, has not been provided yet. Furthermore, the KDG aldolase activity measured in cell extracts of P. torridus and T. acidophilum has not been purified and characterized, in particular with respect to substrate specificity, and the genes encoding these enzymes have not been identified. The biochemical analysis of this aldolase is crucial to define the enzyme as a KDG-specific aldolase, indicative of a nonphosphorylative ED pathway, or as bifunctional KD(P)GA, indicative of the branched ED pathway as proposed for the crenarchaea Sulfolobus and Thermoproteus.In this communication we studied the sugar-degrading pathway in P. torridus by in vivo labeling experiments with [¹³C]glucose, by enzyme measurements, and by characterization of two key enzymes, gluconate dehydratase and KDG aldolase. The data indicate that P. torridus utilizes a strict nonphosphorylative ED pathway, involving a novel KDG-specific aldolase as a key enzyme, and thus exclude the operation of a branched ED pathway, as in crenarchaea involving a bifunctional KD(P)GA as a key enzyme.     

New Pathway for Nonphosphorylated Degradation of Gluconate by Aspergillus niger

A new nonphosphorylative pathway for gluconate degradation was found in extracts of a strain of Aspergillus niger. The findings indicate that gluconate is dehydrated into 2-keto-3-deoxy-gluconate (KDG), which then is cleaved into glyceraldehyde and pyruvate. 6-Phosphogluconate was not degraded under the same conditions. In addition, KDG was formed from glyceraldehyde and pyruvate. Very weak activity was obtained when glyceraldehyde 3-phosphate replaced glyceraldehyde in this reaction.     

Gluconate dehydratase from the promiscuous Entner-Doudoroff pathway in Sulfolobus solfataricus

An investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 degrees C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner-Doudoroff pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose.     

Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Genetic Control of the 2-Keto-3-Deoxy-d-Gluconate Metabolism in Escherichia coli K-12: kdg Regulon

2-Keto-3-deoxy-gluconate (KDG), an intermediate of the hexuronate pathway in Escherichia coli K-12, is utilized as the sole carbon source only in strains derepressed for the specific KDG-uptake system. KDG is metabolized to pyruvate and glyceraldehyde-3-phosphate via the inducible enzymes KDG-kinase and 2-keto-3-deoxy-6-phosphate-gluconate (KDPG) aldolase. However, another inducible pathway, where the KDG is the branch point, has been demonstrated. Genetic studies of the KDG degradative pathway reported in this paper led to the location of KDG kinase-negative and pleiotropic constitutive mutations. The kdgK locus, presumably the structural gene of the kinase, occurs at min 69 and is co-transducible with xyl. The mutants, simultaneously constitutive for the uptake, kinase, and aldolase, define a kdgR locus at min 36 between the co-transducible markers kdgA and oldD. As to the nature of the control exerted by the kdgR product, we have shown the following. (i) Thermosensitive mutants of the kdgR locus are inducible at low temperature but derepressed at 42 C for the three operons—kdgT (transport system), kdgK, and kdgA (KDPG aldolase). (ii) The kdgR⁺ allele is dominant to the kdgR constitutive allele. (iii) A deletion in kdgA extending into the regulatory gene, kdgR, leads to a constitutive expression of the nondeleted operons—kdgT and kdgK. These properties demonstrate that the kdg regulon is negatively controlled by the kdgR product. It is presumed that differences in operator and in promotor structures could explain the strong decoordination, respectively, in the induction and catabolic repression, of these three enzymes activities.     

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.     

Glyceraldehyde dehydrogenases from the thermoacidophilic euryarchaeota Picrophilus torridus and Thermoplasma acidophilum, key enzymes of the non-phosphorylative Entner-Doudoroff pathway, constitute a novel enzyme family within the aldehyde dehydrogenase superfamily

Cells of Picrophilus torridus, grown on glucose, contained all enzyme activities of a non-phosphorylative Entner-Doudoroff pathway, including glucose dehydrogenase, gluconate dehydratase, 2-keto-3-deoxygluconate aldolase, glyceraldehyde dehydrogenase (GADH), glycerate kinase (2-phosphoglycerate forming), enolase and pyruvate kinase. GADH was purified to homogeneity. The 115-kDa homodimeric protein catalyzed the oxidation of glyceraldehyde with NADP+ at highest catalytic efficiency. NAD+ was not used. By MALDI-TOF analysis, open reading frame (ORF) Pto0332 was identified in the genome of P. torridus as the encoding gene, designated gadh, and the recombinant GADH was characterized. In Thermoplasma acidophilum ORF Ta0809 represents a gadh homolog with highest sequence identity; the gene was expressed and the recombinant protein was characterized as functional GADH with properties very similar to the P. torridus enzyme. Sequence comparison and phylogenetic analysis define both GADHs as members of novel enzyme family within the aldehyde dehydrogenase superfamily.     

Identification and characterization of Thermoplasma acidophilum glyceraldehyde dehydrogenase: a new class of NADP+-specific aldehyde dehydrogenase

Thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus solfataricus are known to metabolize D-glucose via the nED (non-phosphorylated Entner-Doudoroff) pathway. In the present study, we identified and characterized a glyceraldehyde dehydrogenase involved in the downstream portion of the nED pathway. This glyceraldehyde dehydrogenase was purified from T. acidophilum cell extracts by sequential chromatography on DEAE-Sepharose, Q-Sepharose, Phenyl-Sepharose and Affi-Gel Blue columns. SDS/PAGE of the purified enzyme showed a molecular mass of approx. 53 kDa, whereas the molecular mass of the native protein was 215 kDa, indicating that glyceraldehyde dehydrogenase is a tetrameric protein. By MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) peptide fingerprinting of the purified protein, it was found that the gene product of Ta0809 in the T. acidophilum genome database corresponds to the purified glyceraldehyde dehydrogenase. The native enzyme showed the highest activity towards glyceraldehyde, but no activity towards aliphatic or aromatic aldehydes, and no activity when NAD+ was substituted for NADP+. Analysis of the amino acid sequence and enzyme inhibition studies indicated that this glyceraldehyde dehydrogenase belongs to the ALDH (aldehyde dehydrogenase) superfamily. BLAST searches showed that homologues of the Ta0809 protein are not present in the Sulfolobus genome. Possible differences between T. acidophilum (Euryarchaeota) and S. solfataricus (Crenarchaeaota) in terms of the glycolytic pathway are thus expected.     

Maltose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the operation of a novel sugar fermentation pathway

The hyperthermophilic anaerobe Pyrococcus furiosus was grown on maltose as energy and carbon source. During growth 1 mol maltose was fermented to 3–4 mol acetate, 6–7 mol H₂ and 3–4 mol CO₂. The presence of the following enzyme activities in cell extracts of maltose-grown P. furiosus indicate that the sugar is degraded to pyruvate and H₂ by a modified non-phosphorylated Entner-Doudoroff-pathway (the values given in brackets are specific enzyme activities at 100 °C): Glucose: methyl viologen oxidoreductase (0.03 U/mg); 2-keto-3-deoxy-gluconate aldolase (0.03 U/mg); glyceraldehyde: benzyl viologen oxidoreductase (2.6 U/mg), glycerate kinase (2-phosphoglycerate forming) (0.48 U/mg), enolase (10.4 U/mg), pyruvate kinase (1.4 U/mg). Hexokinase, glucose-6-phosphate dehydrogenase, 2-keto-3-deoxy-6-phosphogluconate aldolase and phosphofructokinase could not be detected. Further conversion of pyruvate to acetate, CO₂ and H₂ involves pyruvate: ferredoxin oxidoreductase (0.4 U/mg; T=60°C with Clostridium pasteurianum ferredoxin as electron acceptor), hydrogen: methyl viologen ixodoreductase (3.4 U/mg) and ADP-dependent acetyl-CoA synthetase (1.9 U/mg). Phosphate acetyl transferase and acetate kinase could not be detected. The ADP-dependent acetyl-CoA synthetase catalyzes ATP synthesis via the mechanism of substrate level phosphorylation and apparently constitutes the only ATP conserving site during maltose catabolism in P. furiosus.This novel pathway of maltose fermentation to acetate, CO₂ and H₂ in the anaerobic archaeon P. furiosus may represent a phylogenetically ancient pathway of sugar fermentation.Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - BV benzyl viologen - CHES cyclohexylamino-ethane sulfonic acid - ABTS 2,2-Azino-di-(3-ethylbenzthiazoliumsulfonate)     

Properties of enzymes involved in D-galactonate catabolism in fungi

Two enzymes catalyze the two step reactions in the D-galactonate nonphosphorolytic catabolic pathway ofAspergillus terreus, namely D-galactonate dehydratase and 2-keto-3-deoxy-D-galactonate (KDGal) aldolase. Maximum enzyme activities were obtained at 40° C and pH 8.0 or at 50° C and pH 7.5 for these two enzymes, respectively. Stability of the two enzymes under different conditions was investigated. From a Lineweaver-Burk plot of the reciprocal of initial velocities and substrate concentrations, apparent K _m values were calculated for D-galactonate, pyruvate and glyceraldehyde and found to be 8.33, 14.28 and 5.55 mM, respectively, in crude cell-free extracts. Results indicated the requirement of magnesium cation for D-galactonate dehydratase activity at an initial concentration of 10^–2 M. The presence of Mg²⁺ in the reaction mixture seems to induce greatly the fitness of the dehydratase with D-galactonate as no activity could be detected with 24-h dialyzed extract in the absence of magnesium cation.     

Identification and characterization ofThermoplasma acidophilum 2-keto-3-deoxy-D-gluconate kinase: A new class of sugar kinases

The thermoacidophilic archaeonThermoplasma acidophilum has long been known to utilized-glucosevia the non-phosphorylated Entner-Doudoroff (nED) pathway. We now report the identification of a gene encoding 2-keto-3-deoxy-d-gluconate (KDG) kinase. The discovery of this gene implies the presence of a glycolysis pathway, other than the nED pathway. It was found that Ta0122 in theT. acidophilum genome corresponded to KDG kinase. This enzyme shares no similarity with known KDG kinases, and belongs to a novel class of sugar kinases. Of the five sugars tested only KDG was utilized as a substrate.     

Cellobiose catabolism in the haloalkaliphilic hydrolytic bacterium <Emphasis Type="Italic">Alkaliflexus imshenetskii</Emphasis>

Cellobiose metabolism was studied in Alkaliflexus imshenetskii, a haloalkaliphilic hydrolytic bacterium capable of utilizing certain polymers of plant origin, as well as mono- and disaccharides. The major products of cellobiose fermentation by the bacterium were succinate and acetate, and formate was a minor product. Cellobiose could be split into glucose molecules by both β-glucosidase (hydrolytic pathway) and phosphorylase (phosphorolytic pathway); the activity of the former enzyme was two orders of magnitude higher (3600 nmol/(min mg) versus 36 nmol/(min mg)). In cell extracts of the bacterium, high activities of the Embden-Meyerhof-Parnas pathway enzymes—hexokinase, glucose-phosphate isomerase, and phosphofructokinase—were revealed, as well as the activities of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and key enzymes of the Entner-Doudoroff pathway—6-phospho-gluconate dehydratase and 2-keto-3-deoxy-6-phospho-gluconate aldolase. Neither the activity of the key enzyme of the hexose-mono-phosphate pathway, 6-phospho-gluconate dehydrogenase, nor the activities of the key enzymes of the modified Entner-Doudoroff pathway, glucose dehydrogenase and 2-keto-3-deoxy-gluconate kinase, were revealed.     

Glucose metabolism in the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus.

Sulfolobus solfataricus is a thermophilic archaebacterium able to grow at 87 degrees C and pH 3.5 on glucose as sole carbon source. The organism metabolizes glucose by two main routes. The first route involves an ATP-dependent phosphorylation to give glucose 6-phosphate, which readily isomerizes to fructose 6-phosphate. In the second route, glucose is converted into gluconate by an NAD+-dependent dehydrogenation; gluconate is then dehydrated to 2-keto-3-deoxygluconate, which, in turn, is cleaved to pyruvate and glyceraldehyde. Each metabolic step has been tested in vitro at 70 degrees C on dialysed homogenates or partially purified fractions; minimal requirements of single enzymes have been evaluated. Identification of the intermediates is based on chromatographic, spectroscopic and/or synthetic evidence and on specific enzymic assays. The oxidative breakdown of glucose to pyruvate occurring in S. solfataricus differs from the Entner-Doudoroff pattern in that there is an absence of any phosphorylation step.